Evaluation of Caspase‐3 Activity During Apoptosis with Fluorescence Lifetime‐Based Cytometry Measurements and Phasor Analyses

Abstract

Caspase‐3 is a well‐described protease with many roles that impact the fate of a cell. During apoptosis, caspase‐3 acts as an executioner caspase with important proteolytic functions that lead to the final stages of programmed cell death. Owing to this key role, caspase‐3 is exploited intracellularly as a target of control of apoptosis for therapeutic outcomes. Yet the activation of caspase‐3 during apoptosis is challenged by other roles and functions (e.g., paracrine signaling). This brief report presents a way to track caspase‐3 levels using a flow cytometer that measures excited state fluorescence lifetimes and a signal processing approach that leads to a graphical phasor‐based interpretation. An established Förster resonance energy transfer (FRET) bioprobe was used for this test; the connected donor and acceptor fluorophore is cleavable by caspase‐3 during apoptosis induction. With the cell‐by‐cell decay kinetic data and phasor analyses we generate a caspase activation trajectory, which is used to interpret activation throughout apoptosis. When lifetime‐based cytometry is combined with a FRET bioprobe and phasor analyses, enzyme activation can be simplified and quantified with phase and modulation data. We envision extrapolating this approach to high content screening, and reinforce the power of phasor approaches with cytometric data. Analyses such as these can be used to cluster cells by their phase and modulation “lifetime fingerprint” when the intracellular fluorescent probe is utilized as a sensor of enzyme activity. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals LLC on behalf of International Society for Advancement of Cytometry.

A Review of New High-Throughput Methods Designed for Fluorescence Lifetime Sensing From Cells and Tissues

“…The goal of the specialty bioprobes was to enable quantitative analysis of enzyme activity for high-content screening toward the discovery of potent anti-cancer agents. This work was later extended by the inclusion of TRFC phasor analyses, which provided statistical “fingerprints” of the loss of FRET, which was correlated to caspase enzyme activity at the onset of apoptosis [ 34 ]. In other fd-FCM studies, apoptosis-dependent phagocytosis of bacteria-inoculated cells was examined to determine if EGFP lifetimes could be used as a pH indicator, since pH changes are often expected in the phagosome microenvironment during phagocytosis of bacteria cells by macrophages [ 9 ].…”

“…The applications of fluorescence lifetime measurements range significantly. Some examples include separating fluorophores that have similar spectra but different lifetimes [4][5][6][7][8][9][10][11][12][13], exploiting the difference in lifetime of free and bound metabolites for metabolic study and diagnosis [14][15][16][17][18][19][20][21][22][23][24], using FRET to screen for binding or inhibition of exogenous and endogenous molecules of interest [2,5,[25][26][27][28][29][30][31][32][33][34][35], evaluating the conformation and stability of proteins or other molecules in varying environments [31,36], or even using lifetime as an additional parameter in fluorescent inks for anti-counterfeiting [37], though this list is far from exhaustive. New methods and technologies continue to be discovered, further establishing the wide-reaching significance and potential of fluorescence lifetime.…”

Development of Tailor-Made Dendrimer Ternary Complexes for Drug/Gene Co-Delivery in Cancer

“…Both intrinsic and extrinsic pathways induce a set of molecular events that culminate into the activation of caspase-3. Caspase-3 acts as an executioner caspase with an important proteolytic role leading to the final steps of apoptosis [ 62 ].…”

Effect of Chlorhexidine Digluconate in Early Wound Healing of Human Gingival Tissues. A Histological, Immunohistochemical and Biomolecular Analysis

“…Some data reported the centrality of BAX in this pathway, demonstrating that BAX-deficient cells were protected from p53-induced apoptosis [43]. On the other hand, although caspase 3 has been also defined as an enzyme with an important role in the initiation of apoptosis [44], the occurrence of BAX-mediated apoptosis in a caspaseindependent manner has been reported [45]. Therefore, BAX expression seems to be more relevant than caspase 3 activation.…”